Our long term objective is to develop a drug that specifically impedes the growth of cancer cells expressing the EWSR1-FLI1 protein. A strong body of evidence supports the notion that the EWSR1-FLI1 fusion protein exerts its oncogenic action by altering transcription of downstream target genes. Small molecules that inhibit EWSR1- FLI1 function should mimic these transcriptional changes. Using small-molecule-regulated depletion of EWSR1- FLI1, we identified increased DKK1 transcription as a sensitive and specific indicator of EWSR1-FLI1 protein levels (Preliminary Studies, Project I, Aim 3). We have engineered a reporter allele into the endogenous locus of DKK1, in order to determine the impact of a small molecule on DKK1 transcription in high-throughput. Using this system, we have analyzed the impact of 170,000 compounds from the UTSW chemical library leading to the identification of multiple independent scaffolds that, like EWSR1-FLI1 suppression, upregulate DKK1. Here, we propose to screen an additional 160,000 compounds with the aim of advancing one or more to testing in preclinical models of EWS. Candidates will be advanced based on 1) DKK1 upregulation that is verified to be the consequence of EWSR1-FLI suppression, 2) selective anti-proliferative effects on EWS cells, and 3) successful medicinal chemistry efforts to improve potency and pharmacodynamics. These leads will be evaluated for efficacy in a series of in vivo models that include subcutaneous and orthotopic xenograft tumors derived from EWS cancer cell lines as well as patient-derived EWS xenografts. We have also developed the only genetically-engineered model of EWS using zebrafish to provide an autochthonous model for validation. Our team is skilled and experienced in using both chemical proteomics and forward genetics to identify the direct target of anti-cancer small molecules. Concomitant with our evaluation of efficacy, we will identify the direct targets of compounds that inhibit EWSR1-FLI1 with the expectation that an understanding of the mechanism of action will not only unveil biological insight into EWSR1-FLI1 function but also catalyze drug development. Aim 1 is a high throughput screen for small molecule inhibitors EWSR1-FLI1 transcriptional function, based on a nanoluciferase knock-in reporter system. Aim 2 will identify the direct protein target for lead compounds that selectively impede the proliferation of EWS cells. Aim 3 focuses on preclinical advancement of lead molecules, testing efficacy in patient-derived EWS and zebrafish pre-clinical model systems.